Molecular genetic analysis of recombinant mouse strains with crossovers in the H-2 major histocompatibility complex has established that recombination is not random but localized to specific sites which have been termed recombination hot spots. Recombination hot spots map within the Ebeta and Ealpha immune response genes, between the K and Abeta3 genes, and between the Abeta2 and Abeta3 genes. Analysis of recombination hot spots are H-2 haplotype specific and thus are dependent on specific DNA sequences. All of the recombinant mouse strains that have been found to crossover in the Ealpha gene were derived from crosses between strains of mice with Ealpha genes of the K and P haplotypes. The goal of this research project will be to determine the DNA sequences responsible for recombination at this site and to determine how these sequences recognized by enzymes involved in recombination. The Ealpha genes will be cloned from recombinant parental mouse strains and the DNA containing the recombination sites will be sequenced. DNA surrounding the Ealpha recombination hot spot will be analyzed for repeat sequences, deletions, and duplications. DNA sequence of the Ealpha recombination hot spot will be compared to the DNA sequences of the Ebeta and Abeta3 Abeta2 recombination hot spots to determine if there are any sequences in common. Functional studies will be undertaken to examine proteins present during meiosis for ability to interact with DNA fragments containing the Ealpha recombination hot spot. Proteins to be studied include meiosis specific endonucleases, recombinase, and DNA binding proteins. Although recombination hot spots occur in other regions of the mammalian genome such as human insulin and b-globin genes, these hot spots have not been as well characterized as the mouse MHC. The well characterized mouse H-2 complex is the idea system for studying site specific homologous recombination and for determining the mechanisms that direct recombination to these sites.